The present invention relates to photoactivatable therapeutic compounds and compositions, and methods of using said compounds and compositions to inhibit the function and/or expression of targeted DNA. More particularly, the invention disclosed herein relates to the therapeutic uses of photoactivatable therapeutic compositions comprising UVA (ultraviolet A light) irradiation products of nucleotide bases (the oligonucleotides) having predetermined sequences, and with compounds having two photoactivatable functional groups and which are reactable with at least one base of the oligonucleotides under UVA irradiation conditions.
Psoralen compounds are particularly useful as the difunctional compounds in the practice of this invention. The composition is administered to viable cells, preferably in vivo. The cells are thereafter exposed to UVA radiation. This results in inactivated or "turned-off" genes in the cell which have been targeted by the predetermined sequence of the oligonucleotide in the cells.
The oligonucleotide used to prepare the photoactivatable therapeutic material contains a base sequence segment that is complementary to a segment of the "sense" strand or a base sequence segment of the "antisense" DNA strand of a targeted gene. In the preparation of the photoactivatable therapeutic material, it is believed that the oligonucleotide reacts with a photoactivatable moiety of the difunctional compound upon UVA irradiation to form a monoadduct, and upon cellular administration and further UVA irradiation, the photoactivatable therapeutic composition crosslinks in situ to the complementary segment of DNA in the targeted gene.
Disclosed herein are photoactivatable therapeutic drugs which are believed to be chimeric molecules having a photoactivatable moiety and an oligonucleotide moiety, for example, photoactivatable psoralen monoadducts of oligonucleotides; methods for selectively inhibiting DNA function, for example, irradiating the psoralen-oligonucleotide monoadducts in viable cells with UVA; and methods for treating diseases comprising administration of the photoactivatable therapeutic materials of the present invention to targeted tissue, such as skin, followed by localized UVA irradiation of the site.
Phototherapeutic techniques have been known since biblical times. The Egyptians ingested the leaves of a weed (ammi majus) that grew by the Nile and then exposed themselves to sunlight to treat depigmented patches of skin (vitiligo). The active constituent of the ammi majus plant, isolated and characterized in 1947, is a derivative of psoralen, 8-methoxypsoralen (8-MOP). Lerner and associates, in 1953, established that purified 8-MOP could be administered safely to humans and photosensitized 8-MOP was efficacious at low doses in the management of vitiligo. The work by Lerner and associates created great interest and initiated the modern era of therapeutic photopharmacology, particularly in the field of dermatology. Subsequent research demonstrated that UVA irradiated psoralen derivatives (furocoumarins), such as 8-MOP, react with DNA. The therapeutic effects of photoactivated psoralens resulted from their photoreaction with bases in the two complementary strands of DNA.
Phototherapies that have been developed thus far using derivatives of psoralen, such as 8-MOP, have involved UVA light irradiation of skin after topical application or oral ingestion of the psoralen derivative and, more recently, extracorporeal irradiation of diseased blood.
Two of the earliest phototherapies were the treatment of vitiligo and psoriasis with psoralen and UVA. A more recent development has been the treatment of the leukemic phase of cutaneous T-cell lymphoma (CTCL) with psoralen and UVA. After oral administration of 8-MOP, a patient's blood is separated into three fractions by leukophoresis: erythrocytes, leukocytes and plasma. The leukocytes and plasma are combined and passed through a plastic cassette in which they are irradiated as a thin (1 mm) layer. "Treatment of Cutaneous T-Cell Lymphoma by Extracorporeal Photochemotherapy", Edelson, et al., Vol. 316, No. 6, The New England Jour. Med., pp. 297-303, Feb. 5, 1987.
The psoralen derivative, 8-MOP (I), is a naturally occurring tricyclic aromatic compound whose planar structure facilitates intercalation between nucleic acid base pairs in DNA. ##STR1##
When activated with UVA light (320-400 nm), psoralens react with pyrimidine bases, preferentially with thymine, in cellular DNA. The preference for thymine over other nucleotide bases is about 20:1. Psoralens are polyfunctional reagents which form three distinct types of photoadducts with DNA: two monoaddition products and one cross-linked product. The monoadducts are either 3,4-"pyrone-side" cyclobutyl adducts of psoralen with the 5,6 carbons of thymine (such as illustrated for thymine above, II), or 4',5'-"furan-side" cyclobutyl adducts of psoralen with thymine (such as illustrated for thymine above, III). ##STR2##
The 3,4-monoadduct of psoralen (II) has only a weak absorption band near 300 nm and therefore only weakly absorbs UVA light. The 4',5'-monoadduct of psoralen has an intense absorption band near 330 nm and thus is more readily capable of absorbing a second UVA photon. The second photon photoactivates the 3,4-pyrone ring leading to a second reaction with another thymine base (such as illustrated for thymine above, IV) on the opposite DNA strand thereby producing crosslinked DNA strands. In other words a psoralen acts to crosslink DNA strands by reacting with thymine bases in DNA strands. The 4',5'-monoadduct absorbs another UVA photon. Thus, crosslink formation of DNA is a two-photon process in which the absorption of the first photon leads to the formation of the 4',5'-monoadduct of psoralen to DNA, and the absorption of the second photon of UVA photoactivates the 3,4-pyrone ring of psoralen which subsequently adds to another thymine on the opposite DNA strand to give the crosslinked DNA. ##STR3##
Base pairing between complementary nucleic acid strands provides hydrophobic sites which are exquisitely suited for occupation by nonpolar psoralen molecules. Photoactivation of the bound and oriented psoralen moieties promotes the formation of psoralen photoadducts with pyrimidine bases (primarily thymine) in DNA. As noted above, possible reaction products include monoaddition adducts (monoadducts) and one diadduct or crosslink.
Several factors affect the photocondensation process. The ability of a particular psoralen to interact with DNA depends on its intrinsic thermodynamic properties which are related to the nature of the substituents on the core psoralen molecule. The psoralen partitions itself between the aqueous environment surrounding the DNA and the hydrophobic sites between base pairs. The distribution can be shifted towards more base pair intercalation by a) lowering the temperature, b) increasing the salt concentration of the reaction buffer, and c) altering substituents on the psoralen core (e.g., 4'-aminomethyl-4'5',8-trimethylpsoralen (AMT) in place of 8-MOP).
In the absence of base pairs the yield of psoralen photoadducts is reduced, and in the case of 8-MDP the yield is reduced by at least two orders of magnitude. Therefore base paired strands of DNA are used in the preferred method of producing the monoadducts of this invention.
To control the formation of monoadducts two strategies have been used. First, by irradiating psoralen-double stranded DNA solutions with wavelengths of UV light greater than 390 nm, monoadducts are virtually the only photoadducts formed. Second, by intentionally incorporating a mismatched base in the AT region of the oligonucleotide in the strand complementary to the target strand, monoadducts alone are produced regardless of which UVA wavelength is used for irradiation. For example, structure (V) represents a matched base pair, and structure (VI) represents a mismatched base pair wherein the mismatched complementary strand has an A in place of the complementary base T, thereby inhibiting psoralen from crosslinking between T bases in the mismatched strands.
______________________________________ GAGTATGAG (V) CATAC GAGTATGAG (VI) CAAAC ______________________________________
By intentionally incorporating a mismatched base in the complementary strand, crosslink formation is blocked. Following irradiation, the mixture containing the monoadduct oligonucleotide is subject to denaturation to separate the unreacted complementary DNA strand from the oligonucleotide-psoralen monoadduct strand. Preliminary results indicate that monoadducts form with similar efficiencies when 8-MOP plus ds-DNA, either perfectly matched or containing a single mis-matched base, are irradiated with 400 nm light.
Phototherapies of the prior art involve the administration of a derivative of psoralen, for example 8-MOP, and subsequent UVA irradiation to produce monoadducts and crosslinks in complementary strands of DNA in irradiated cells that contain the 8-MOP. However, administration of a psoralen compound by itself is not specific for any particular locus of the DNA contained in the cells, and subsequent UVA irradiation causes the formation of monoadducts and crosslinks randomly throughout the cellular DNA.
One of the objects of the present invention is to target a particular nucleotide sequence in the cellular gene for photo-crosslinking with a monoadduct of a predetermined sequence of oligonucleotides and a compound that has at least two photoactivatable functional groups, which compound is reactable with at least one base of the oligonucleotide under UVA irradiation conditions. The result of such crosslinking reaction is that the gene containing the reaction product is turned off, i.e., the crosslinked DNA is inhibited from replicating and expressing the polypeptide for which it codes. Thus, at least one targeted segment of DNA of an entire genome is selectively modified by a crosslink in that gene, or some other DNA segment. Other genomic loci are therefore spared the effects of the photoactivated compound, such as psoralen. Oligomers complementary to a segment of one strand of DNA in the targeted gene, which are capable of locating and hybridizing with the targeted segment, were designed as the means of delivering photoactivatable compounds, such as the psoralen-oligonucleotide adduct, to a specific nucleotide sequence in the cellular gene prior to UVA irradiation and crosslink formation.
Although we do not wish to be bound by any theory by which inactivation of a targeted gene occurs, we believe that a photoactivated reaction occurs in vivo resulting in a gene or genes that are inactivated or "turned off". Thus, the therapeutic materials of this invention are referred to as photoactivatable because their therapeutic effects are initiated by UVA. It is believed that in the initial reaction, in vitro, a psoralen molecule conjugates to an oligonucleotide to form the 4',5'-monoadduct of the psoralen and the oligonucleotide.
The psoralen-oligonucleotide monoadducts of the present invention are administered to viable cells or tissue, such as by injection, or topical or intralesional application to the skin of animals, and are transported or otherwise caused to enter cells. We theorize that in the cell, the psoralen-oligonucleotide adduct hybridizes with the specific or complementary segment of DNA in the targeted DNA sequence. Subsequent in situ UVA irradiation of the hybridized photoactivatable psoralen-oligonucleotide material causes the psoralen-oligonucleotide material to react with or crosslink to the complementary strand to which it is hybridized and thereby inactivates the targeted DNA sequence.
Once the DNA sequence of a gene is known, or once the amino acid sequence of a polypeptide for which the gene codes are known, specific oligonucleotide sequences can be constructed by methods known in the art. The specific oligonucleotide sequence that is constructed should be complementary to at least a segment of DNA of the gene that is to be turned off. Therefore, any known gene locus containing a TA sequence or AT sequence would be a potential target for the phototherapeutic materials of this invention because the psoralen moiety of the monoadduct preferentially reacts with thymine bases, both in the oligonucleotide and in the cellular DNA.
The compositions of the present invention comprise photoactivatable oligonucleotides, e.g., the 4',5'-monoadducts of a psoralen compound, with specific, predetermined oligonucleotides that contain at least a TA or AT sequence and desirably a TAT or ATA sequence. These chimeric 4',5'-monoadduct compounds are believed to be formed after UVA irradiation of a mixture of a psoralen and a oligonucleotide of a predetermined sequence. The 4',5'-monoadduct of the psoralen may be separated from the reaction mixture that may contain other photoadducts produced by this first irradiation, as well as unreacted materials.
Other compounds containing at least two photoactivatable functional groups, which compounds are reactable with at least one base of the oligonucleotide under UVA irradiation conditions and therefore are usable in the therapeutic treatments described herein, include: N,N'-alkylene(C-1 to C-15)-bis-maleimides (e.g., N,N'-tetramethylene-bis-maleimide); N-H or N-alkyl(C-1 or C-8)-furanocarbostyrils (e.g., 7H-furano[3,2-g]quinolone-2); furanochromones (e.g., 7H-furano [3,2-g][1]benzopyran-5-one); and alkenyl(C-3 to C-10)oxycoumarins (e.g., 7-allyloxycoumarin).
The oligonucleotide moiety of the monoadduct is designed to hybridize to a segment of DNA of the targeted gene that contains a thymine base within the complementary sequence of the DNA. Thus, the useful irradiated product contains a psoralen 4',5'-monoadduct at a thymine site in the predetermined sequence of the oligonucleotide. The 4',5'-monoadduct is capable of hybridizing with and thereafter photoreacting with a complementary DNA sequence in a targeted gene.
Further, oligodeoxyribonucleotide- and oligodeoxyribonucleoside-alkylphosphonates complementary to selected sequences in targeted genes, are within the meaning of oligonucleotides or oligomers as used herein for the practice of the disclosed invention. Oligomers of oligodeoxyribonucleoside-lower(C-1 to C-3)alkylphosphonates are nonionic nucleic acid analogues that contain a neutral alkylphosphonate linkage that replaces the negatively charged phosphodiester internucleotide bond normally found in nucleic acids. The phosphonate linkage is resistant to hydrolysis by nucleases and therefore the nonionic oligonucleotides are able to penetrate cell walls. The invention contemplates that all or some of the internucleotide phosphates may be replaced with alkyl phosphonates. Selective replacement of the internucleotide phosphates with an alkyl (e.g., methyl) phosphonate to form a "hybrid" molecule has been found to be particularly advantageous in suppressing the activity of nucleolytic enzymes located in the skin toward these modified oligonucleotides. Significantly lesser concentration of oligonucleotide having a hybrid internucleotide linkage may be needed to achieve the same cellular effect as compared to completely substituted oligonucleotides.
The oligonucleotides used in this invention comprise predetermined sequences of DNA bases ranging in size from about 8 bases up to about 30 bases. Most preferably, the sequence is between about 12 and about 20 bases in length. Less than 8 bases in the sequence may be used as the predetermined sequence, however, the degree of uniqueness decreases rapidly with decreasing length thereby greatly reducing the potential specificity of the psoralen-oligonucleotide monoadduct for a targeted gene. In other words, the psoralen-oligonucleotide monoadduct may interact with other genes (genes not intended as the target gene) and cause undesirable inactivation of such other genes. On the other hand predetermined oligonucleotide sequences greater than about 30 bases may demand such a high degree of homology for recognition and hybridization of its complementary segment in the gene to be targeted, that it may prevent practical application of this invention. In other words, the time that may be required for hybridization of the longer predetermined oligonucleotide sequences to the complementary DNA segments of the targeted gene may render this process ineffective as a practical matter. Moreover, the transport across cell walls of 4',5'-psoralen-olignucleotide monoadducts having a long predetermined oligonucleotide sequence may be retarded or even inhibited.
With respect to the predetermined oligonucleotide sequence, the sequence must contain at least a thymine-adenine or adenine-thymine (TA or AT) dinucleotide sequence within the oligonucleotide. In other words, the predetermined sequence is selected from a complementary segment of DNA of the gene to be targeted that contains an AT or TA dinucleotide sequence. Preferably, the predetermined oligonucleotide sequence that is selected should contain multiple TA or AT sequences within the predetermined sequence. For example, TAT or ATA, and TATA or ATAT are preferred. It is also possible, but less desirable, for a cytosine to replace a thymine in the oligonucleotide or the target gene.
Accordingly, the predetermined sequence that is selected and has, for example 10 bases, may contain a TATA sequence and 6 additional bases. For example, in the gene for T3 (T cell receptor), the sequence 5'-GGAATATAGG-3' is found and it is appreciated that the selected sequence contains the desirable ATATA sequence. That 10 base sequence may be selected as the predetermined complimentary oligonucleotide sequence for use in this invention. In this example, the complementary oligonucleotide sequence normally is 5'-CCTATATTCC-3'.
The oligonucleotide sequence may be prepared by techniques well known in the art. For example, the oligonucleotide sequence may be prepared by solid phase synthesis in a DNA synthesizer, or less preferred, the complex technique of reverse transcriptase may be used.
In order to prepare the psoralen-oligonucleotide monoadduct, a buffered (pH 7.4) aqueous solution of psoralen, 0.05-0.2 mg/ml, is prepared. For each ml of psoralen solution there is added about 10-50 micrograms of the predetermined oligonucleotide at a concentration of about 0.01-0.05 mM. Thereafter the solution is irradiated with long wavelength UVA light (390-410 nm) from a monochromator for about 1-5 hours for each ml of psoralen-oligonucleotide solution. Larger solutions may be used if one irradiates for longer periods of time. In place of a UV lamp, one may use an incandescent or special fluorescent lamp. A UV lamp could be used together with a glass filter that eliminates the UVA wavelength that cause the crosslinking reaction, namely 320-370 nm.
The method of the present invention comprises administering to viable cells a therapeutically effective amount of the photoactivatable oligonucleotide, such as the 4',5'-psoralen-oligonucleotide monoadduct. Hybridization of the oligonucleotide moiety to its complementary DNA genetic locus in the cell is believed to occur and the cell is then irradiated with UVA light. The hybridized 4',5'-psoralen-oligonucleotide monoadduct crosslinks to the complementary DNA sequence within the targeted gene. Photoactivation of the 3,4-pyrone ring of the psoralen moiety of the psoralen-oligonucleotide monoadduct, and its subsequent addition to a thymine on the targeted DNA to which the oligonucleotide moiety of the monoadduct is hybridized, provides a diadduct crosslink thereby inhibiting the replication and expression of the genetic information in the targeted gene.
There are perhaps 1,000 diseases and disorders of the skin. The skin is the body's largest organ, amounting to 15 percent of the total body weight. Within each centimeter, there are about 100 sweat glands, four meters of nerves and more than 3,000 nerve cells responsive to touch, temperature, pressure and pain. It is also a vital part of the body's immune system; the highest concentration of lymphocytes involved in the body's defense mechanism is located in the skin. Further, epidermal cells are known to produce significant amounts of interleukin-1, interleukin-3, thymopoietin, interferon, granulocyte/monocyte colony stimulating factor, lymphocyte function-associated antigens (e.g., LFA-1) and intercellular adhesion molecules (e.g., ICAM-1). Inhibiting these genes or inhibiting other genes that encode proteins or peptides, such as CD1a, that participate in the immune reaction, from expressing such polypeptide products allows one to effectively treat a variety of serious or troublesome disorders of the immune system.
By examining the nucleotide sequence of any of the genes responsible for the production of the aforementioned polypeptides, or for any other targeted gene, one can select and prepare complementary oligonucleotides to specific AT or TA containing 8-30 base sequences within the targeted gene. The complementary sequences are used to prepare the compositions of the present invention. For example, the following sequences are complementary to sequences in the gene that encodes the intracellular adhesion molecule ICAM-1:
(1) TTT AGG CAA CGG GGT CTC TAT GCC CAA CAA CTT GGG CTG, PA0 (2) CTC GCT CTG GTT CCC CAG TAT TAC TGC ACA CGT CAG CCG, PA0 (3) CCG GGT CTG GTT CTT GTG TAT AAG CTG GCC GGC CAC CTC and PA0 (4) GAG GCC TGC AGT GCC CAT TAT GAC TGC GGC TGC TAC CAC. PA0 (1) TGT CAC CAA CCT CCA ACT TAT TCA CCT TCC CCT AAT TC, PA0 (2) CA TAT C ATT TGC AGA TGT TAT TTC CTT CTC TCA GAA AAA, PA0 (3) GAA GTA GCA AAA ACA GCA TAT CAT TTG CAG ATG TTA TTT, PA0 (4) TGG AAT TGC TGT CCC AGG TAT GAG TCT GCA AAT CAC TCA, PA0 (5) AAA TGA CCG AAT GGT GCG TAT ACG GAA TAA TGT TTC CAG, PA0 (6) ACA GCC TCC TGT CAC CTG TAT CTC AAA AGG ATC TGG CCT, PA0 (7) ATA TTC CCA GCC ACT GGA TAT GGC AAC CAT GAA TTG TTC, PA0 (8) TGC AGA AAT GCT TGG CCA TAT TCC CAG CCA CTG GAT ATG, PA0 (9) AAG AAG TAA XXX AGG CAC TAT CAC CGC CAA GAT GAT GAA, PA0 (10) AAC TGC AAT TCA TCG GCG TAT CTA CGA ATT CCC TCA AAT, PA0 (11) ACC TGT ATC TCA AAA GGA TAT TCA AAC TGC AAT TCA TGG, PA0 (12) ACA GCC TCC TGT CAC CTG TAT CTC AAA AGG ATA TTC AAA, PA0 (13) ATA TTC CCA GCC ACT GGA TAT GGC AAC CAT GAA TTG TTC, and PA0 (14) ACT GAG AAG ATT GTG TGT TAT GTC ATT TTC ATG CTG ATT, PA0 (1) GGC GAC GCA CAT GGA CAC TAT GTA GAA AGA GCT GTC TCC, PA0 (2) TTT GAC CGT TCA GCC CGA TAT CTG AGC TCA AAG CGT AGT, PA0 (3) GAA TAT TAT CAT CGT CTT TAT TAG TAG TAA GTG CCT GCA, PA0 (4) AAT CTC TGA AGA GAA TAT TAT CAT CGT CTT TAT TAG TAG, PA0 (5) TGG GGA AGA AGT AGT CTG TAT TGC TGA TGT CAT AAG GGC and PA0 (6) TGG TGC CAC CCA GCC AGC TAT CTG GGG AAG AAC TAG TCT. PA0 (1) GAA AGC CTT GCA AGA GGC TAT AAG CAG CCC TGC AGG GCA, PA0 (2) CTG CTA CAG AGG AAT GGA TAT AGA GAT CTT GAC TAC CCA, PA0 (3) ATA CCC TCT GTG CCC CTG TAT AAT CAA TAC CTT CTC TCC, PA0 (4) TTC TGT GTG GGG AAG CAC TAT TTC AAA AGC CCC TCT GTG, PA0 (5) CCG TAG ACC CTG CTC GAA TAT CTT CAG GCG GGT CTG CAC, PA0 (6) ACC GGA GTT GGG GGG CAG TAT GTC TGG TAG TAG CTG GCT, PA0 (7) CTA GGG CTG AAT AGG AGC TAT GGC CTG TTC TTG GGG GGC, and PA0 (8) CGT GGG GAA AGA ACT GTG TAT TTC TCT CTC GCT GCT GAG. PA0 (1) ATG ATC CTC ATA AAG TTG TAT TTC ACA TTG CTC AGG AAG PA0 (2) CTG ATC ATT GGC TCG AAT TAT ACT TTG ATT GAG GGG GTC PA0 (3) ACC TGT GAT GGT TTT GGG TAT CTC AGG CAT CTC CTT CAG PA0 (4) CTA CGC CTG GTT TTC CAG TAT CTG AAA GTC AGT GAT AGA
As it is most preferable that the oligonucleotide used in the compositions according to the invention contain an ATA or TAT sequence and be 12 to 20 nucleotide bases in length, several sequences included within each of the above sequences could be particularly suitable for producing a composition that inhibits expression of ICAM-1.
Similarly, the following sequences are complementary to sequences in the gene that encodes CD1a(T-6):
wherein X=A,T,G or C.
Included within these sequences are 12 to 20 base sequences containing an AT or TA, and most preferably a TAT or ATA sequence that would be particularly useful in preparing compositions according to the present invention that could be used to block CD1a production.
Several sequences complementary to regions in the gene that encodes IL-6 receptor protein could be useful in blocking expression of the protein. These include the following:
TA or AT-containing, or most preferably TAT-containing 12 to 20 base sequences that are included within these sequences can be used to produce compositions according to the invention for inhibiting expression of the IL-6 receptor protein.
Oligonucleotides complementary to regions in the gene that encodes GM-CSF include those having the following sequences:
Photoactivatable compositions prepared using 12-20 base TA or AT-containing, and most preferably ATA or TAT-containing, oligonucleotides having sequences that are contained within these sequences can be used to inhibit expression of GM-CSF.
The following sequences are complementary to regions in the gene encoding IL-1.alpha.:
AT or TA-containing, or preferably ATA or TAT-containing oligonucleotides having sequences contained within these sequences are useful in preparing compositions according to the present invention for inhibition of IL-1.alpha. expression.
The compositions and methods of the present invention are particularly suitable for treatment of skin disorders by local applications, such as topical application to skin or injection into the tissue to be treated with the photoactivatable therapeutic composition, because of the ease of subjecting epidermal cells to UVA irradiation. The photoactivatable therapeutic composition permeates the outer layer of the skin. Viable cells in the lower layer of the epidermis absorb the photoactivatable material into the cellular nuclei where the above referred to hybridization and subsequent UVA irradiation takes place.
Among the diseases which can be treated by the compositions of this invention are inflammatory diseases (such as atopic dermatis or lupus erythematosus), diseases of keratinization (such as ichthyosis or psoriasis), viral diseases (such as warts and herpes simplex), and neoplastic diseases (such as melanoma and cutaneous T cell lymphoma).
In addition, the photoactivatable oligonucleotide compositions and methods of the present invention can be effective in the selective inhibition of not only endogenous genes, but also exogenous genes, such as those derived from viral genomes present in skin cells or other targeted cells which are capable of being UVA irradiated.
The monoadducted oligonucleotides described herein may be formulated in suitable pharmaceutical vehicles for topical or intralesional administration for treatment of skin disorders, such as psoriasis. The instant compositions can be applied topically to or injected into the treatment site, e.g., subcutaneously by injection. When used for topical applications, the adduct is usually formulated with a pharmaceutically acceptable carrier.
Carrier materials are well known in the pharmaceutical formulation art and include those materials referred to as diluents or vehicles. The carrier may include inorganic or organic materials and should have sufficient viscosity to allow spreading of the composition and provide good adherence to the tissue to which it is topically applied. Examples of such carriers include, without limitation, polyols such as glycerol, propylene glycol, polyethylene glycol, suitable mixtures thereof, vegetable oils, and other materials well known to those skilled in this art. The viscosity of the formulation can be adjusted by methods well known in the art, for example, by the use of a higher molecular weight polyethylene glycols.
In addition to the adduct and carrier, the formulation may contain pharmacologically-acceptable additives or adjuvants such as antimicrobial agents, e.g., methyl, ethyl, propyl, and butyl esters of para-hydroxybenzoic acid, as well as chlorobutanol, phenol, ascorbic acid, etc. The formulation may also contain thickening or gelling agents, emulsifiers, wetting agents, coloring agents, buffers, stabilizers and preservatives including antioxidants such as butylhydroxyanisole in accordance with the practice of the art. The formulation may also contain penetration enhancers such as dimethyl sulfoxide, long-chain alcohols such as nonoxynol, long-chain carboxylic acids, propylene glycol, N-(2-hydroxyethyl)pyrrolidone,l-dodecyl-azacycloheptan-2-one, and the like. Depending on the method of application and specific therapeutic requirements, it may be desirable to use absorption-delaying agents such as aluminum monostearate and gelatin.
The composition of the formulation may be adjusted using components well-known in the formulation art to provide a pharmaceutical formulation which is a gel, cream, ointment, solid, liquid, semi-solid, etc. The particular physical form of the formulation depends on the desired method of treatment and the patient to be treated.
Typical formulations of the pharmaceutical compositions of this invention are set forth as follows:
______________________________________ Application Form Formulation (Per 100 gms.) ______________________________________ Cream Monoadducted (preferred range Oligonucleotide 0.01-10%) Ascorbic acid 0.1 Benzyl alcohol 1 Propylene glycol 15 Water 25 Stearyl alcohol 6 Cetyl alcohol 5 White petrolatum 15 Poloxyl-40 stearate 8 Injectable Monoadducted (preferred range Liquid Oligonucleotide 0.01-10%) Water 35 Glycerine 5 Sodium chloride 1 Sodium ascorbate 0.1 Propylene glycol 5 ______________________________________
For administration by injection, the compositions according to the invention are formulated as solutions or suspensions having a low enough viscosity to be injected. The composition suitable for injectable use must be sterile and fluid to the extent that it allows easy injection. It should also be stable under conditions of manufacture and storage and be preserved against contamination by microorganisms. Preservatives include alcohol, benzoic acid, sorbic acid, and methyl and propyl paraben with and without propylene glycol. Additionally, the pH of the composition must be within the range which does not result in tissue damage or lead to chemical instability of the adduct, namely, between about 4-7.
The concentrations of active ingredients in a particular formulation required to provide a particular effective dose may be determined by a person skilled in the pharmaceutical formulation art based upon the properties of a carrier and the particular additives introduced into the formulation. Formulations may be prepared that have significantly higher concentrations of adduct depending upon the carrier and additives being used. If the carrier substantially retains the adduct or releases it at a slow rate, the concentrations of the adduct in the formulation can be substantially increased and in fact may have to be substantially increased in order to provide an effective treatment. In practice, it is preferred that a formulation contain the lowest concentrations of adduct which effectively treat the condition with the desired number of applications, i.e., a lower effective dose rate can be tolerated if multiple applications are used. This low concentration limit is dependent upon the delivery effectiveness of the carrier vehicle. Preferably, the adduct comprises between about 0.01 and about 10 weight percent of the formulation.
A preferred embodiment of the instant invention comprises formulations containing adduct, i.e., oligonucleotide 8MOP monoadduct. This formulation is particularly effective in treating psoriasis. Although the effective concentration of adduct delivered to the treatment site depends, inter alia, upon the carrier and other additives included in the formulation, ordinarily the concentration of adduct in the formulation ranges from about 0.01 to 10 weight percent. These ranges are provided by way of description and not by way of limitation since it is recognized that the concentration may be adjusted over a wide range depending on the carrier material, number of applications used, etc., as described hereinabove.
The pH of the formulation is important in assuring stability of the adduct as well as assuring that the formulation is physiologically acceptable to the patient. The pH of the formulation may be maintained through the use of toxicologically acceptable buffers. Such buffers are well known in the pharmaceutical formulation art, and include hydrochloric acid buffer, acid phthalate buffer, phosphate buffer and citric acid/sodium citrate buffer.
In topical applications the instant compositions are applied to the affected area or afflicted situs for the patient. The term "topical" refers herein to the surface of the epidermal tissue, especially the skin, and surface of psoriatic disease on the skin which have been modified, as well as sites from which plaques have been removed from the skin.
In preparing a formulation suitable for topical application, the adduct is normally mixed with a suitable solvent. Examples of solvents which are effective for this purpose include ethanol, acetone, acetic acid, aqueous alkaline solutions, dimethyl sulfoxide, glycerine, glycerol, propylene glycol, nonoxynol, ethyl ether, polyethylene glycol, etc.
Application by injection can be used for treatment of psoriasis. In this procedure the instant composition is injected directly into the psoriatically involved skin.